Glycoproteins are proteins that have one or more covalently attached sugar polymers. N-linked protein glycosylation is an essential and conserved process occurring in the endoplasmic reticulum of eukaryotic organisms. It is important for protein folding, oligomerization, stability, quality control, sorting and transport of secretory and membrane proteins (Helenius. A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019-1049).
Protein glycosylation has a profound influence on the immunogenicity, the stability and the half-life of a protein. In addition, glycosylation can assist the purification of proteins by chromatography, e.g. affinity chromatography with lectin ligands bound to a solid phase interacting with glycosylated moieties of the protein. It is therefore established practice to produce many glycosylated proteins recombinantly in eukaryotic cells to provide biologically and pharmaceutically useful glycosylation patterns.
WO 200307467 (Aebi et al.) demonstrated that the food-borne pathogen Campylobacter jejuni, which is a bacterium, could N-glycosylate its proteins, which was a unique feature among known prokaryotic organisms except for certain species of archaea. The machinery required for glycosylation is encoded by 12 genes that are clustered in the so-called pgl locus. Disruption of N-glycosylation affects invasion and pathogenesis of C. jejuni but is not lethal as in most eukaryotic organisms (Burda P. and M. Aebi, (1999). The dolichol pathway of N-linked glycosylation. Biochem Biophys Acta 1426(2):239-57). It is possible to reconstitute the N-glycosylation of C. jejuni proteins by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli the same time (Wacker et al. (2002). N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790-1793).
N-glycans have a glycan attached to a consensus sequence in a protein. The known N-glycosylation consensus sequence in a protein allows for the N-glycosylation of recombinant target proteins in prokaryotic organisms. Such organisms comprise an oligosaccharyl transferase (“OT”; “OTase”), such as, for example, an oligosaccharyl transferase of C. jejuni, which is an enzyme that transfers the glycan to the consensus sequence of the protein.
WO 200307467 (Aebi et al.) teaches a prokaryotic organism into which is introduced a nucleic acid encoding for (i) specific glycosyltransferases for the assembly of an oligosaccharide on a lipid carrier, (ii) a recombinant target protein comprising a consensus sequence “N-X-S/T”, wherein X can be any amino acid except proline, and (iii) an oligosaccharyl transferase, such as, for example, an oligosaccharyl transferase of C. jejuni that covalently links said oligosaccharide to the consensus sequence of the target protein. Said prokaryotic Organism produces N-glycans with a specific structure which is defined by the type of the specific glycosyltransferases.
WO 2006/119987 (Aebi et al.) describes proteins, as well as means and methods for producing proteins, with efficiency for N-glycosylation in prokaryotic organisms in vivo. It further describes an efficient introduction of N-glycans into recombinant proteins for modifying immunogenicity, stability, biological, prophylactic and/or therapeutic activity of said proteins, and the provision of a host cell that efficiently displays recombinant N-glycosylated proteins of the present invention on its surface. In addition, it describes a recombinant N-glycosylated protein comprising one or more of the following N-glycosylated optimized amino acid sequence(s):
D/E-X-N-Z-S/T (optimized consensus sequence),
wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated partial amino acid sequence(s) is introduced. The introduction of specific partial amino acid sequence(s) (optimized consensus sequence(s)) into proteins leads to proteins that are efficiently N-glycosylated by an oligosaccharyl transferase in these introduced positions.
The biosynthesis of different polysaccharides is conserved in bacterial cells. The polysaccharides are assembled on carrier lipids from common precursors (activated sugar nucleotides) at the cytoplasmic membrane by different glycosyltransferases with defined specificity. Lipopolysaccharides (“LPS”) are provided in gram-negative bacteria only, e.g. Shigella spp., Pseudomonas spp. and E. coli (ExPEC, EHEC).
The synthesis of LPS starts with the addition of a monosaccharide to the carrier lipid undecaprenyl phosphate (“Und-P-P”) at the cytoplasmic side of the membrane. The antigen is built up by sequential addition of monosaccharides from activated sugar nucleotides by different glycosyltransferases, and the lipid-linked polysaccharide is flipped through the membrane by a flippase. The antigen-repeating unit is polymerized by an enzymatic reaction. The polysaccharide is then transferred to the Lipid A by the Ligase WaaL forming the LPS that is exported to the surface, whereas the capsular polysaccharide is released from the carrier lipid after polymerization and exported to the surface. The biosynthetic pathway of these polysaccharides enables the production of LPS bioconjugates in vivo, capturing the polysaccharides in the periplasm to a protein carrier.
Such synthesized complexes of oligo- or polysaccharides (i.e., sugar residues) and proteins (i.e., protein carriers) can be used as conjugate vaccines to protect against a number of bacterial infections. Conjugate vaccines have been successfully used to protect against bacterial infections. The conjugation of an antigenic polysaccharide to a protein carrier is required for protective memory response, as polysaccharides are T-cell independent immunogens. Polysaccharides have been conjugated to protein carriers by different chemical methods, using activation reactive groups in the polysaccharide as well as the protein carrier.
Conjugate vaccines can be administered to children to protect against bacterial infections and also can provide a long lasting immune response to adults. Constructs of WO 2009/04074 (Fernandez, et al.) have been found to generate an IgG response in animals. It has been found that an IgG response to a Shigella O-specific polysaccharide-protein conjugate vaccine in humans correlates with immune protection in humans. (Passwell, J. H. et al., “Safety and Immunogenicity of Improved Shigella O-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel” Infection and Immunity, 69(3):1351-1357 (March 2001).) It is believed that the polysaccharide (i.e. sugar residues) triggers a short-term immune response that is sugar-specific. Indeed, the human immune system generates a strong response to specific polysaccharide surface structures of bacteria, such as O-antigens and capsular polysaccharides. However, since the immune response to polysaccharides is IgM dependent, the immune system develops no memory. The protein carrier that carries the polysaccharide triggers an IgG response that is T-cell dependent and that provides long lasting protection since the immune system develops memory.
E. coli O157 is an enterohemorrhagic strain responsible for approximately two-thirds of all recent cases of hemolytic-uremic syndrome and poses serious human health concerns (Law, D. (2000) J. App. Microbiol., 88, 729-745; Wang, L., and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551).
Escherichia coli strain O157 produces an O-antigen containing the repeating tetrasaccharide unit (4-N-acetyl perosamine→fucose→glucose→GalNAc) (α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc) (Perry, M. B., MacLean, L. and Griffith, D. W. (1986) Biochem. Cell. Biol., 64, 21-28). The tetrasaccharide is preassembled on undecaprenyl pyrophosphate. The E. coli cell envelope contains an inner plasma membrane, a stress-hearing peptidoglycan layer and an asymmetric outer membrane consisting of a phospholipid inner monolayer and an outer monolayer composed of bacterial LPS. LPS contains three components, the lipid A anchor, the 3-deoxy-D-manno-oct-2-ulosonic acid-containing core, and the O-antigen region (see: Raetz, C. R. H. and Whitfield, C. (2002) Annu. Rev. Biochem., 71, 635-700; Whitfield, C. (2006) Ann. Rev. Biochem. 75, 39-68; Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; and refs, therein for reviews on the assembly of O-antigens of bacterial LPS).
The O-antigen components of bacterial LPS are large, extremely diverse polysaccharides that can be either homopolymeric, composed of a single repeating monosaccharide, or heteropolymeric, containing 10-30 repeats of 3-6 sugar units (Reeves, P. R., Hobbs, M., Valvano, M. A., Skurnik, M., Whitfield, C., Coplin, D., Kido, N., Klena, J., Maskell, D., Raetz, C. R. H., and Rick, P. D. (1996) Trends Microbial., 4, 495-503). O-Antigens are, Thus, the Dominant Feature of the bacterial cell surface and constitute important determinants of virulence and pathogenicity (Law, D. (2000) J. App. Microbiol., 88, 729-745; Spears, K. J., Roe, A. J. and Golly, D. L. (2006) FEMS Microbiol. Lett., 255, 187-202; Liu, B., Knirel, Y. A., Feng, L., Perepelov, A. V., Senchenkova, S. N., Wang, Q., Reeves, P. R. and Wang, L (2008) FEMS Microbiol. Rev. 32, 627-653; Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403). E. coli strains with more than 180 individual O-serotypes, attributed to unique O-antigen structures, have been identified (Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403).
O-antigen repeat units are pre-assembled on the cytosolic face of the inner membrane attached to undecaprenyl pyrophosphate. The lipid-linked repeat units diffuse transversely (flip-flop) to the periplasmic surface of the inner membrane and are polymerized before transport to the outer membrane and ligation to LPS. Most heteropolymeric O-antigen repeat units have either N-acetylglucosamine (“GlcNAc”) or N-acetylgalactosamine (“GalNAc”) at the reducing terminus.
It had been assumed that the biosynthesis of the lipid intermediates is initiated by the transfer of GlcNAc-9 or GalNAc-P from their respective sugar nucleotide derivatives to undecaprenyl monophosphate (“Und-P”) catalyzed by WecA (Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; Alexander, D. C. and Valvano, M. A. (1994) J. Bacteriol., 176, 7079-7084; Zhang, L., Radziejewska-Lebrecht, J., Krajewska-Pietrasik, D., Tolvanen, P. and Skurkik. M. (1997) Mol. Microbiol. 23, 63-76; Amor, P. A. and Whitfield, C. (1997) Mol. Microbiol. 26 (145-161); Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Although the properties and specificity of the GlcNAc-phosphotransferase activity of WecA have been characterized (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322), the conclusion that WecA catalyzes the synthesis of GalNAc-P-P-Und was based on genetic studies (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Such earlier genetic studies indicated that the biosynthesis of the lipid-linked tetrasaccharide intermediate was initiated by the enzymatic transfer of GalNAc-P from UDP-GalNAc to Und-P catalyzed by WecA (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). However, there was no direct enzymological evidence demonstrating that WecA utilizes UDP-GalNAc as a GalNAc-P donor.
Furthermore, the E. coli O55 gne and gne1 genes were previously proposed to encode a UDP-GlcNAc 4-epimerase (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625; Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609). Previous reports identified two genes from E. coli O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) and E. coli O86 (Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609), E. coli O55 gne and E. coli O86 gne1, respectively, that are 100% identical to a Z3206 gene within the same gene family.
Accordingly, one of skill would have been led to believe that the Z3206 gene also encodes a UDP-GlcNAc/UDP-GalNAc epimerase.